Project Summary Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb). TB requires a lengthy multidrug treatment and remains difficult to treat because there is considerable drug tolerance among Mtb in the host. To rationally design drug regimens for tuberculosis, we need to understand how Mtb creates and maintains a population structure that generates individuals with diverse drug sensitivities. Mycobacteria exhibit cell-to-cell heterogeneity in fundamental features of their cell physiologies, arising from a deterministic, asymmetric growth and division pattern. This unique growth pattern creates variation in cell size, growth rate, and partitioning of cellular components. Mycobacterial cell size provides critical insight into cell physiology because cell size is tightly connected to antibiotic sensitivity. Mtb alter their cell size distributions under different environmental stressors that are encountered in the host. Despite the key role of stresses during the interactions of pathogenic Mtb and the host, studies of single-cell growth, cell cycle progression, cell size control, and drug susceptibility have been conducted in non-pathogenic, non-Mtb mycobacteria under nutrient-replete growth conditions. A lack of understanding of the details involved in environment-specific control of Mtb cell size and cell population structure is a critical experimental gap that must be bridged to enter into a new phase of designing TB therapies that target drug tolerant subpopulations. To bridge this gap, we seek to understand how Mtb cell growth and replication processes are mediated in various environmental conditions encountered in host tissues and how these characteristics determine antibiotic susceptibility. A systematic, quantitative approach is key to understanding how mycobacteria tolerate antibiotic stress and will allow us to rationally design more effective TB regimens. In this project, we will investigate the process by which Mtb adapt cell size control to different growth environments encountered during host infection and quantitatively characterize in detail the distinct subpopulations of drug-tolerant Mtb. We will use a combination of live-cell microscopy, fluorescent markers, and image analysis. We will integrate these quantitative analyses into mathematical models to rigorously test cellular strategies of cell growth and division. Our multidisciplinary approach will quantify the relationships between cell size and cell cycle progression with drug susceptibility in distinct elemental stress conditions of the host environment. To connect Mtb growth features and variation to treatment outcome in humans, our experiments will focus on clinical isolates from patients that were cured or relapsed. We anticipate that these models will form the basis from which to create optimized treatment regimens that target emerging drug-tolerant subpopulations using different combinations of existing antibiotics.